During the last 3 years we have isolated and partially purified seven species of phosphotyrosyl-casein phosphatase. The activities can be distinguished from one another by their behavior on ion exchange columns and by their apparent molecular weights on gel filtration. We have purified the major phosphotyrosyl- casein phosphatase activity, PTP-5, to near homogeneity and found that it is a monomeric protein (Mr 46,000). We have discovered two heat-stable proteins in bovine brair extracts, termed PTPI-I and PTPI-II, which rather specifically inhibit PTP- 5. The two inhibitor proteins have been partially purified and characterized. Heat-stable inhibitor activity with properties similar to the bovine brain inhibitors was also found to be present at high levels in the brain, kidney and spleen of rabbit. These results suggest that the two inhibitors of PTP-5 may play a physiological role in the regulation of PTP-5. We have also identified a new in vitro substrate for pp60v-src. The substrate is protein phosphatase 1, a major serine- and threonine-specific protein phosphatase. The phosphorylation of protein phosphatase 1 is remarkable because its stoichiometry of phosphorylation (0.34 mol/mol) is significantly higher than that of any other substrate for pp60v-src with the exception of the insulin receptor and because phosphorylation of protein phosphatase 1 decreases its activity by increasing the apparent km for substrate proteins. The phosphorylation of protein phosphatase 1, if it occurs in intact cells, may account in part for the increases in the phosphorylation of proteins on serine and threonine residues observed after transformation of cells with Rous sarcoma virus, which expresses pp60v-src. The experiments in the present proposal are focused on the regulation of PTP-5 by PTPI-I and PTPI-II. The major goals are: 1) to purify and characterized the two heat-stable inhibitor proteins, 2) to develop antibodies to PTP-5, PTPI-I and PTPI-II and 3) to use the pure inhibitor proteins and the three antibodies as powerful new tools to study the role of PTPI-I and PTPI-II in the regulation of PTP-5 and the relationship between PTP-5 and other phosphotyrosyl-protein phosphatases. We will also use protein phosphatase 1 as a substrate to further characterize phosphotyrosyl-protein phosphatase activities in bovine brain extracts.